High Level of Interaction Between Phages and Bacteria in an Artisanal
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bioRxiv preprint doi: https://doi.org/10.1101/2021.08.03.454940; this version posted August 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 High Level of Interaction between Phages and Bacteria in an 2 Artisanal Raw Milk Cheese Microbial Community 3 Luciano Lopes Queiroz1,2, Gustavo Augusto Lacorte2,3, William Ricardo Isidorio2, Mariza 4 Landgraf2, Bernadette Dora Gombossy de Melo Franco2, Uelinton Manoel Pinto2, Christian 5 Hoffmann2* 6 1 Microbiology Graduate Program, Department of Microbiology, Institute of Biomedical 7 Science, University of São Paulo, São Paulo, SP, Brazil 8 2 Food Research Center, Department of Food Sciences and Experimental Nutrition, Faculty 9 of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP, Brazil 10 3 Instituto Federal de Minas Gerais - Campus Bambuí, Bambuí, MG, Brazil 11 *e-mail: [email protected] 12 Abstract 13 Endogenous starter cultures are used in the production of several cheeses around the world, 14 such as Parmigiano-Reggiano, in Italy, Époisses, in France, and Canastra, in Brazil. These 15 microbial communities are responsible for many of the intrinsic characteristics of each of 16 these cheeses. Bacteriophages are ubiquitous around the world, well known to be involved 17 in the modulation of complex microbiological processes. However, little is known about 18 phage–bacteria growth dynamics in cheese production systems, where phages are normally 19 treated as problems, as the viral infections can negatively affect or even eliminate the starter 20 culture during production. Furthermore, a recent metagenomic based meta-analysis has 21 reported that cheeses contain a high abundance of phage-associated sequences. Here, we 22 analyse the viral and bacterial metagenomes of Canastra cheese, a tradition artisanal 23 cheese produced using an endogenous starter culture. We observe a very high phage 24 diversity level, mostly composed of novel sequences. We detect several metagenomic 25 assembled bacterial genomes at strain level resolution, and several putative phage-bacteria 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.03.454940; this version posted August 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 26 interactions, evidenced by the recovered viral and bacterial genomic signatures. We 27 postulate that at least one bacterial strain detected could be endogenous to the Canastra 28 region, in Brazil, and that its growth seems to be modulated by native phages present in this 29 artisanal production system. This relationship is likely to influence the fermentation dynamics 30 and ultimately the sensorial profile of these cheeses, with implication for all cheeses that 31 employ similar production processes around the world. 32 Introduction 33 Viruses are abundant in all ecosystems on Earth, presenting high genetic and taxonomic 34 diversities, shaping biogeochemical cycles and ecosystem dynamics1,2. These obligate 35 intracellular parasites are called bacteriophages (or simply phages) when they infect 36 bacteria. The diversity and composition of phage-bacterial communities are influenced by 37 environmental (e.g. pH, temperature, salinity) and biological factors3,4. Biological factors can 38 be divided in host traits (species abundance, organismal size, distribution, physiological 39 status, and host range) and virus traits (infection type, virion size, burst size, and latent 40 period)5. 41 Many raw milk cheeses around the world are produced using endogenous starter cultures6, 42 a complex microbial community composed of yeasts, bacteria and phage, all of which 43 interact to create the final food product. This procedure often uses the backsloping method, 44 where residual fermented whey is collected during production to be reinoculated as a starter 45 culture on the next day’s production batch, effectively creating a continuous microbial growth 46 system7. Brazil produces a wide variety of artisanal cheeses, several of which use the 47 backsloping method8,9. Canastra cheese is one such cheese, and its endogenous starter 48 cultures have recently been characterized using molecular techniques. They contain a 49 diverse, but stable microbial community10. The community stability in these endogenous 50 starters is assumed to be maintained by a continuous diversification process: when one 51 species is excluded, the genetic function is kept present in the environment by another 52 closely related species11. Studies of Canastra cheese microbiome are usually focused on the 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.03.454940; this version posted August 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 53 bacterial and fungal components12,13; however, little is known about its virome composition 54 and interactions with the bacterial hosts. 55 Most bacteriophages found in dairy fermentation environments belong to the Siphoviridae 56 family, such as 936, P335 and c2 groups. These non-enveloped viruses possess 57 icosahedral morphology and non-contractile tails, with their genome encoded in double- 58 stranded DNA (dsDNA), and commonly infect bacteria from the Lactococcus genus. Other 59 less abundant phage groups are also able to infect bacteria from the genera Leuconostoc, 60 Lactobacillus, Streptococcus, Bacillus, Staphylococcus and Listeria14,15. Phage-bacteria 61 interactions tend to be highly specific, due to phage infection strategies that depend on the 62 host binding proteins and the antiphage defense systems in the host15,16. These defense 63 systems are classified in adaptive immune systems, including several types of CRISPR-Cas 64 systems, and innate immune systems, such as restriction-modification (RM), abortive 65 infection (Abi)17 and, most recently described systems BREX for Bacteriophage Exclusion18 66 and DISARM for Defense Island System Associated with Restriction-Modification19. 67 Here, we present the first description of the virome composition in Brazilian artisanal 68 Canastra cheese and the phage-bacterial interactions in this food system. We identified 69 1,234 viral operational taxonomic units (vOTU) and explored the interactions with bacteria 70 across seven cheese producing properties using a combination of viral and microbial 71 metagenomic sequencing. We characterized a putative novel species of Streptococcus 72 phage 987 group, as well as its potential host, a metagenome assembled genome (MAG) 73 classified as Streptococcus salivarius. Finally, the relationships between 15 complete and 74 high-quality phage genomes and 16 MAGs obtained from starter cultures and cheeses were 75 evaluated. 76 Results 77 VLP-based description of the bacteriophage community present in the Canastra 78 Cheese endogenous starter culture. We assessed the bacteriophage community present 79 in the endogenous starter cultures used by seven artisanal Canastra Cheese producers in 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.03.454940; this version posted August 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 80 Brazil, located in São Roque de Minas and Medeiros, Minas Gerais, Brazil. Viral-like 81 particles (VLPs) were enriched from these starter cultures using a 100 kDa filter membrane 82 and used for metagenome sequencing. The sequencing reads were assembled and 83 rigorously curated to remove bacterial DNA contaminants, producing a final viral sequence 84 catalog (Fig. S1). Our final catalog yielded 908 complete and partial viral genomes, with 85 contig sizes ranging from 1 × 103 bp to 1.7 × 105 bp and the coverage between 1.41 - 86 21108x with genomes classified as: complete (5), high-quality (12), medium-quality (23), 87 low-quality (584), and not-determined quality (284) (Table S1). 88 Family level taxonomic classification for the viral catalog was made using the Demovir 89 pipeline. The order Caudovirales (99%) prevailed, with only a minor number of sequences 90 classified as Algavirales (0.55%) and Imitervirales (0.33%) (Table S1). Contigs were 91 classified at family level as Siphoviridae (43.1%), followed by Myoviridae (12.1%), 92 Podoviridae (3.4%), Phycodnaviridae (0.55%), Mimiviridae (0.33%), and Retroviridae 93 (0.11%). The unassigned contigs corresponded to 40.3% (Fig. 1a). Integrase or site-specific 94 recombinase genes were detected in 67 viral contigs (Fig. 1b) and VirSorter identified 7.38% 95 of this viral sequence catalog as temperate phages (Fig. S2). 96 Alpha diversity analysis of viral metagenomes was calculated using a normalized count table 97 of reads mapped against the viral contigs. Four out of seven samples showed high values of 98 diversity index (> 6 Shannon and > 0.94 Simpson), one sample showed medium values 99 (4.66 Shannon and 0.78 Simpson), and two samples, low values (< 3 Shannon and < 0.6 100 Simpson) (Table S2). The sample with lowest diversity values (P43 sample, 1.29 Shannon 101 and 0.39 Simpson) was dominated by one complete, high coverage viral genome (>21000x) 102 belonging to the Siphoviridae family (Fig. 1c), and 94 contigs were classified at species level 103 (Table S1; Supplementary Results) 104 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.08.03.454940; this version posted August 3, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.